A review of the toxicology and pathology of the gastrointestinal tract.

The gastrointestinal tract of man and animals shows great specialization in structure and function for its primary role of digestion. There are many species differences in diet, anatomy and metabolism, and its neuroendocrine regulation has evolved into a complex field for investigation. Exposure of the tract from oral cavity, stomach, small and large intestine results in a range of toxicities covered by this review. Carcinogenesis of the gastrointestinal tract by a range of agents including pharmaceuticals is also discussed.

Cell-mediated responses in dogs with spontaneous neoplasms. I. Detection of cell-mediated cytotoxicity by the chromium-51 release assay.

A series of 43 dogs with spontaneous melanomas, sarcomas, or mammary carcinomas were tested for peripheral blood lymphocytotoxicity against a range of allogeneic tumor cells in vitro on one or more occasions during therapy. All tumor-bearer groups contained a proportion of dogs showing a significant 51Cr release with increasing effector-to-target cell ratios. However, cytotoxicity was not restricted for target cells of the same histologic type as that of the effector cell donor. Some unrelated target cells more sensitive to nonspecific effects showed a greater cytotoxicity in some instances. Control effector cells from healthy dogs were nonspecifically cytotoxic for a range of tumor target cells in a similar proportion of instances. Short-term cultures of autochthonous tumor target cells were resistant to lysis in the 51Cr release assay. The findings did not provide evidence for the existence of specific tumor antigens operative to allogeneic 51Cr release cytotoxicity assays.

Cell-mediated responses in dogs with spontaneous neoplasms. II. Detection by direct leukocyte migration inhibition technique.

A series of 39 dogs bearing spontaneous mammary carcinomas or melanomas were tested for tumor-directed immune response against allogeneic Formalin-treated tumor cells. The immune response was assessed by the direct leukocyte migration inhibition technique. Comparison of leukocytes from tumor bearers and healthy donors by chi-square analysis showed no statistical difference between the groups. The findings did not provide evidence for the existence of specific tumor antigens in the dog.

Biomarkers of drug-induced vascular injury.

In pre-clinical safety studies, drug-induced vascular injury is an issue of concern because there are no obvious diagnostic markers for pre-clinical or clinical monitoring and there is an intellectual gap in our understanding of the pathogenesis of this lesion. While vasodilatation and increased shear stress appear to play a role, the exact mechanism(s) of injury to the primary targets, smooth muscle and endothelial cells are unknown. However, evaluation of novel markers for potential clinical monitoring with a mechanistic underpinning would add value in risk assessment and management. This mini review focuses on the progress to identify diagnostic markers of drug-induced vascular injury. Von Willebrand factor (vWF), released upon perturbation of endothelial cells, is transiently increased in plasma prior to morphological evidence of damage in dogs or rats treated with vascular toxicants. Therefore, vWF might be a predictive biomarker of vascular injury. However, vWF is not an appropriate biomarker of lesion progression or severity since levels return to baseline values when there is morphological evidence of injury. A potential mechanistically linked biomarker of vascular injury is caveolin-1. Expression of this protein, localized primarily to smooth muscle and endothelial cells, decreases with the onset of vascular damage. Since vascular injury involves multiple mediators and cell types, evaluation of a panel rather than a single biomarker may be more useful in monitoring early and severe progressive vascular injury.

Mitogenic responses of canine peripheral blood lymphocytes to staphylococcal protein A.

In order to produce long term lymphoid cell cultures from canine lymphocytes of known histocompatibility antigen specificities, mitogenic responses to staphylococcal protein A (SpA) were examined and compared with those of phytohaemagglutinin (PHA) and concanavalin A (Con A). SpA was found to be the strongest mitogen tested with significant responses to concentrations as low as 31 ng/ml. There was a decrease in responsiveness above optimal mitogen concentrations with SpA and PHA. Peak responses were observed at lower concentrations for longer incubation times. PHA showed a rapid fall off in thymidine uptake below optimal concentrations whereas the SpA dose-response curve was less steep and a shoulder or secondary peak of activity was observed at low SpA concentrations in some cases. Continuous SpA stimulation of lymphocyte cultures resulted in an initial period of cell proliferation followed usually by a second period of cell proliferation around week 7 of culture. To date, viable cell cultures have been maintained for up to 12 weeks in vitro. SpA lymphoblast cultures behave normally in microcytotoxicity tests for serologically defined DLA histocompatibility antigens and remain functional in natural killer (NK) and PHA induced cell mediated cytotoxic reactions against 51Cr-labelled tumour target cells but were not themselves susceptible as target cells for NK activity.

The predictivity of the toxicity of pharmaceuticals in humans from animal data--an interim assessment.

This project was undertaken by the International Life Sciences Institute's Health and Environmental Sciences Institute (ILSI-HESI) to develop an improved understanding of the extent to which various types of human toxicities (HTs) manifested during clinical trials could be predicted from standard toxicology studies. A multi-company database of 131 pharmaceutical agents to-date was based on compounds with one or more demonstrated HTs identified during clinical development. These interim results support a true positive prediction rate of animal models for human toxicity of 69%, and also that study results from non-rodent (dog, primate) species have good potential to identify HTs from many therapeutic classes. The continuing assessment of a larger database may have impact on the identification of new toxicology methodologies, and may lead to optimization of non-clinical study designs and improved assessments.

Phenobarbitone-induced liver response in wild type and in p53 deficient mice.

The tumour suppressor protein, p53, is involved in the regulation of apoptosis and growth arrest following DNA damage. Mutations of the p53 gene are found in 50-55% of all human cancers (Hollstein et al. Nucl. Acid Res. 22 (1994) 3551), including hepatocellular carcinomas. Phenobarbitone (PB) is a non-genotoxic hepatocarcinogen in rats and mice. With commercial availability of mice where one or both alleles of p53 have been removed we have examined the effect of PB in wild type C57BL/6J mice (p53 +/+), and p53 deficient mice (+/- and -/- p53) to determine whether p53 plays a role in the PB induced liver response. In each strain of mice, chronic administration caused liver enlargement, which was associated with centrilobular hepatocyte hypertrophy and a transient hyperplasia. In addition, an increase in centrilobular epidermal growth factor receptor and its ligand, transforming growth factor alpha and a decrease in mannose-6-phosphate receptor and its mitoinhibitory ligand, TGFbeta1 was also observed immunohistochemically. The similar response in all three strains indicates that p53 probably plays no role in the early PB induced liver effects of hypertrophy and changes in growth factor expression.

Toxic effects of the histamine H(2) receptor antagonist metiamide on bone-marrow haemopoietic and stromal progenitor cells in vitro.

The histamine H(2)-receptor antagonist, metiamide has been shown to cause agranulocytosis in vivo. In vitro colony forming assays for bone-marrow stromal fibroblast progenitors (CFU-F) and granulocyte/macrophage progenitor cells (CFU-GM) were performed, using murine bone marrow, to assess the relative sensitivity of committed haemopoietic cells, and the marrow stromal microenvironment to metiamide toxicity. CFU-F were more susceptible than CFU-GM to inhibition by metiamide, with 50% inhibition of colony formation (ID(50)) at 17 and 180 mug/ml in the CFU-F and CFU-GM assays, respectively. Inhibition of CFU-GM required the continuous presence of the drug, while CFU-F were inhibited similarly by either short-term (20-hr) or prolonged (10-day) incubation with metiamide (ID(50) 27 and 17 mug/ml, respectively). It is suggested that bone-marrow stromal cell damage may be an important contributory factor in the haemopoietic toxicity of metiamide.

An overview of animal toxicology studies with bicalutamide (ICI 176,334).

The toxicological profile of bicalutamide in animals following acute and chronic dosing is closely associated with the drug's non-steroidal anti-androgenic pharmacological activity. Bicalutamide produces typical effects of an anti-androgen, including atrophy of the prostate, testis and seminal vesicles and Leydig cell hyperplasia resulting from inhibition of pituitary feedback by testosterone. Subsequent benign Leydig cell tumors were seen in rats, but Leydig cell hyperplasia has not been observed in patients. Bicalutamide causes liver enlargement and is a mixed function oxidase inducer in rodents and dogs, but not man. These effects lead to thyroid hypertrophy and adenoma in the rat and hepatocellular carcinoma in the male mouse. In vitro and in vivo genotoxicity studies have all given negative results. Bicalutamide also caused a reversible shortening of the electrocardiographic P-R interval in the dog without any associated pathology. This change was not detected in ECG monitoring during clinical trials. In conclusion, bicalutamide produced a range of pharmacological effects, as well as liver enlargement with enzyme induction and dog ECG changes in preclinical toxicity studies in rodents and dogs. Only the pharmacological changes were found to be relevant to human usage.

A critical review of the optimum duration of chronic rodent testing for the determination of non-tumourigenic toxic potential: a report by the BTS Working Party on Duration of Toxicity Testing.

This review indicates that for the detection of non-neoplastic toxic effects: 1. Four decades of accumulated literature provide no lead as to the optimum duration of repeat dose toxicity testing required for all classes of chemicals, although 6 months repeated administration appears adequate for pharmaceuticals. 2. Three month studies predicted the 2 year outcome for 70% of the compounds evaluated in this pilot study using data published by the US National Toxicology Program. 3. In spite of the limitations of this pilot study, this finding is considered encouraging as it is close to that generated previously on more detailed confidential pharmaceutical data. This suggests that the exercise should now be expanded using confidential surveys of industrial data to determine the concordance resulting from the evaluation of a larger group of chemicals.

Prospects for reducing and refining the use of dogs in the regulatory toxicity testing of pharmaceuticals.

A workshop was held to critically discuss the need for a nonrodent species and the role of the dog in regulatory toxicity testing of pharmaceuticals; to discuss opportunities to reduce and refine the use of dogs in preclinical toxicology; and to identify a number of specific recommendations which could be feasibly achieved to move the process forward. To facilitate a preliminary evaluation of the contribution of dog studies to the risk assessment process, anonymised, unpublished data were provided from fully evaluated, repeat-dose toxicity studies in the rat and dog. Results of the International Life Sciences Institute (ILSI) Human Toxicity Project were also presented and discussed. Analysis of the data demonstrated that the dog can provide additional toxicity information, which, in some cases, was shown to be predictive for humans. Discussions indicated that there is potential for achieving a reduction in dog use and several possible approaches were identified. To further the progress of this initiative, there is a need to collate the results of pharmacology, toxicology, and clinical studies to address some of the proposed approaches. One of the outcomes of the workshop will be the establishment of a steering group to co-ordinate data collation for further analysis.

Pathology of the forestomach in rats treated for 1 year with a new histamine H2-receptor antagonist, SK&F 93479 trihydrochloride.

The new long acting histamine H2-receptor antagonist was administered to rats at doses of 1000, 200 and 40 mg/kg body weight. Forestomach lesions of marked focal hyperplasia and hyperkeratosis were observed at a 48% incidence after 1 year daily gavage dosing with 1000 mg/kg. Lesions of this type were not seen in mid- and low-dose groups. The basal epithelium was convoluted and showed frequent mitotic figures. Two cases (4%) showed hyperplastic epithelium penetrating the muscularis mucosae. Recovery high-dose animals left untreated for a further 20-22 weeks were examined by endoscopy and necropsy and showed the hyperplasia and associated hyperkeratosis to be reversible. As penetration of the muscularis mucosae was only present in two cases at 1 year, the reversibility of this stage could not be assessed and the significance of the lesion will be determined by the results of a 2 year carcinogenicity study.

Agglutination reactions of spontaneous canine tumour cells, induced by concanavalin A, demonstrated by an isotopic assay.

Quantitative assessment of the agglutination of 51Cr labelled canine cell suspensions to canine kidney cell monolayers has been performed over a range of concanavalin A concentrations. Agglutination was observed with all cell cultures tested, comprising four spontaneous canine melanomas, two canine mammary carcinomas, a benign mammary tumour and a contact-inhibited kidney cell line. The melanomas tested showed strong specific inhibition of concanavalin A agglutination by 10(-2)M alpha-methyl-D-glucopyranoside. Inhibition of agglutination of mammary tumour and kidney cells was weaker and less specific. Agglutination was inhibited at 4degrees C. Reduced agglutination to glutaraldehyde-fixed mono-layers was observed in the case of mammary tumours but was absent when contact-inhibited kidney cells were tested. The specificity of the reaction for transformed cells and the parameters involved are discussed.

Spontaneous neoplasms of the marmoset (Callithrix jacchus). Oral and nasopharyngeal squamous cell carcinomas.

Eight spontaneous highly invasive oral and nasopharyngeal squamous cell carcinomas were observed over an 18-month period in a breeding colony of the common marmoset (Callithrix jacchus). Affected marmosets were predominantly males over four years of age. The incidence of this tumor in the four-year-plus age group was 4.9%. The tumors were locally invasive through the palate to the nasal cavity, retrobulbar space and cranial cavity in some marmosets with lung metastases present in three cases.

Allogeneic grafts of spontaneous canine melanomas and their cell culture strains in neonatal immunosuppressed dogs.

Canine melanoma has been transplanted to allogeneic neonatal recipients receiving continuous immunosuppression with anti-lymphocyte serum. One spontaneous melanoma was directly transplanted into 8 recipients, 6 of which developed tumours. 5/5 melanoma cell cultures were transplantable, with 19 tumour takes in 31 allogeneic recipients. Serial passage was performed in the case of two melanomas. Tumour development required continuous immunosuppression and the site was dependent upon the route of inoculation and other factors. Transplanted cell cultures were all amelanotic in vitro and in vivo, except in the case of one melanoma which reverted to a melanotic morphology after in vivo growth.

Coronary vascular lesions in dogs treated with phosphodiesterase III inhibitors.

A treatment-related coronary arteriopathy has been observed in the dog following the oral or intravenous administration of 4 potent phosphodiesterase type III inhibiting inodilators at high multiples of their ED50 for periods from 1 day to 6 months. A fifth compound of a similar pharmacological class exhibited limiting toxicity at low multiples of its ED50 and this compound failed to induce coronary arterial lesions. The earliest treatment-related findings observed were medial hemorrhage and necrosis with focal breaks in the internal elastic lamina. Later changes, observed from day 9 onwards, included intimal thickening consisting of smooth muscle proliferation with a mucoid ground substance, variable and inconsistent inflammatory changes involving one or more arterial tunics and adventitial hemorrhage, fibrosis and neovascularization. The changes were restricted to the coronary arteries including the extramural and intramural branches. The distribution of lesions varied from widespread, multifocal involvement of both coronary arterial systems to focal lesions with no obvious site of predilection. Induction of this lesion may involve changes in coronary flow and pressure as a result of an exaggerated pharmacological response to this class of compound. The susceptibility of other species (rat, cynomolgus monkey, or pig) to this effect has been investigated with no treatment-related arteriopathy being observed.

Secondary responses of alloantigen-primed dog lymphocytes.

The usefulness of Primed Lymphocyte Typing (PLT) for recognizing Lymphocyte Defined (LD) determinants of the Major Histocompatibility Complex of the dog (DLA) was assessed in 10 separate experiments. Application of a technique described for human cell to peripheral blood lymphocytes of dogs gave reproducible, informative results. DLA LD determinants were shown to be of major importance in secondary responses of alloantigen "primed" lymphocytes, but other, a yet undefined, factors also appeared to play a role in the assay.

Gastric neuroendocrine cell hyperplasia after treatment with the long-acting, potent H2-receptor antagonist SK&F 93479.

The time course and dose response of the neuroendocrine cell hyperplasia in the oxyntic mucosa of the rat was examined after treatment with the potent, long-acting H2-receptor antagonist SK&F 93479 at doses of 0 and 1000 mg/kg orally for 1, 3, 7, and 14 days and at doses of 0, 40, 200, and 1000 mg/kg orally for 1 and 6 months. The number of oxyntic neuroendocrine cells (chromogranin-positive) increased after 7 days of treatment. In the 1- and 6-month studies with doses of 1000 mg/kg, the grading for the number of oxyntic chromogranin-positive cells was 2.5 to 3 times the control levels, and they were distributed mostly throughout the mucosa, whereas at lower doses, which did not produce carcinoid tumours at 2 years, the neuroendocrine cells were distributed in the lower half of the mucosa with 1.5- to 2-fold increases in grades for cell numbers. Increases in cell numbers and cell distribution may be useful factors in the evaluation of the neuroendocrine cell hyperplasia found in, for example, the Zollinger-Ellison syndrome and chronic atrophic gastritis, in which hypergastrinaemia and fundic neuroendocrine cell hyperplasia are present.

Comparative toxicology of temelastine. A novel H1 antagonist in dog, rat, and monkey.

The toxicity of temelastine 2-[4-(5-bromo-3-methylpyrid-2-yl)butylamino]-5-[(6-methylpyrid+ ++-3-yl) methyl]-4-pyrimidone a potent, selective, competitive histamine H1-receptor antagonist was examined in dogs and rats. The major toxicological response seen in the dog was marked, but intermittent and reversible, increases in the plasma activity of a number of liver-associated enzymes, viz alanine aminotransferase (ALT), glutamate dehydrogenase (GLDH), and alkaline phosphatase (ALP). The increases first seen in two male dogs treated for 30 consecutive days at a dose of 300 mg/kg became apparent at lower doses, i.e., 100 and 33.3 mg/kg/day, in 6- and 12-month studies. Although the increases were suggestive of hepatotoxicity, the only histological changes were increases in hepatocellular lipofuscin pigment and foci of macrophages seen in dogs treated at 300 mg/kg for 12 months. Rats treated for up to 12 months at doses as high as 300 mg/kg/day showed no treatment-related increases in plasma enzymes although increases in liver weights and hepatocellular lipofuscin pigment together with centrilobular hypertrophy were seen in the 300 mg/kg/day treatment group. To investigate differences in hepatic responsiveness between species dogs, rats, and monkeys were exposed to high concentrations of temelastine by continuous 24-hr intravenous infusion. The results of the study showed the dog to be most sensitive to the hepatic effects of temelastine. The major toxicological effect of temelastine in the rat was a histopathological lesion of the thyroid gland characterized by agglomeration and depletion of colloid, follicular epithelial hypertrophy and reduced follicular size. The no-effect dose for this lesion was between 10 and 33.3 mg/kg/day. These histopathological changes, characteristic of a "TSH-driven" thyroid gland, were not seen in the thyroid glands of dogs.